Synthesis of the cyclic and acyclic acetal derivatives of 1-(3-C-Ethynyl-β-D-ribo-pentofuranosyl)cytosine, a potent antitumor nucleoside. Design of prodrugs to be selectively activated in tumor tissues via the bio-Reduction-Hydrolysis mechanism

Article abstract of DOI:10.1016/S0968-0896(03)00104-4

We have designed and synthesized the acetal derivatives of 1-(3-C-ethynyl-β-D-ribo-pentofuranosyl)cytosine (ECyd, 1), the 2′,3′-O-nitrobenzylidene derivatives 2 and 3 and the 5′-O-(alkoxy)(nitrophenyl)methyl derivatives 6-10 as potential prodrugs of ECyd. These prodrugs can be selectively activated in tumor tissues via a bio-reduction-hydrolysis mechanism owing to the characteristic properties of tumor tissues, such as hypoxia and lower pH. Although the 2′,3′-O-(4-nitrobenzylidene) derivatives 2 and 3 were converted bio-reductively into the corresponding 4-aminobenzylidene derivatives by rat S-9 mix, the reduction products, that is, the corresponding amino congeners 4 and 5, proved to be rather stable in an aqueous solution at pH 6.5 used as a pH model for acidic tumor tissues. In contrast, the 5′-O-(alkoxy)(4-nitropheny)methyl derivatives 6-8 were also reduced by rat S-9 mix to the corresponding amino congeners 11-13, which were hydrolyzed to release ECyd more effectively at pH 6.5 than at pH 7.4. Accordingly, the acyclic acetals 6-8 may be efficient prodrugs of ECyd, that are effectively reduced under physiological conditions releasing ECyd in acidic tumor tissues.

Full text of DOI:10.1016/S0968-0896(03)00104-4

Bioorganic & Medicinal Chemistry 11 (2003) 2453–2461
Synthesis of the Cyclic and Acyclic Acetal Derivatives of
1-(3-C-Ethynyl-ꢀ-D-ribo-pentofuranosyl)cytosine, a
Potent Antitumor Nucleoside.Design of Prodrugs
to be Selectively Activated in Tumor Tissues Via
the Bio-Reduction–Hydrolysis Mechanismy
Makoto Nomura,a,b Satoshi Shutoa and Akira Matsudaa,*
aGraduate School of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812, Japan
bHanno Research Center, Taiho Pharmaceutical Co. Ltd., 1-27 Misugidai, Hanno, Saitama 357-8527, Japan
Received 9 January 2003; accepted 6February 2003
Abstract—We have designed and synthesized the acetal derivatives of 1-(3-C-ethynyl-b-d-ribo-pentofuranosyl)cytosine (ECyd, 1),
the 20,30-O-nitrobenzylidene derivatives 2 and 3 and the 50-O-(alkoxy)(nitrophenyl)methyl derivatives 6–10 as potential prodrugs of
ECyd. These prodrugs can be selectively activated in tumor tissues via a bio-reduction–hydrolysis mechanism owing to the char-
acteristic properties of tumor tissues, such as hypoxia and lower pH. Although the 20,30-O-(4-nitrobenzylidene) derivatives 2 and 3
were converted bio-reductively into the corresponding 4-aminobenzylidene derivatives by rat S-9 mix, the reduction products, that
is, the corresponding amino congeners 4 and 5, proved to be rather stable in an aqueous solution at pH 6.5 used as a pH model for
acidic tumor tissues. In contrast, the 50-O-(alkoxy)(4-nitropheny)methyl derivatives 6–8 were also reduced by rat S-9 mix to the
corresponding amino congeners 11–13, which were hydrolyzed to release ECyd more effectively at pH 6.5 than at pH 7.4.
Accordingly, the acyclic acetals 6–8 may be efficient prodrugs of ECyd, that are effectively reduced under physiological conditions
releasing ECyd in acidic tumor tissues.
# 2003 Elsevier Science Ltd. All rights reserved.
Introduction
1)1 to be a potent antitumor nucleoside that significantly
inhibits the growth of various human solid tumor cells
both in vitro and in vivo. It is now in clinical trials.
Studies on the antitumor mechanism of action of ECyd
showed that after being metabolized to its 50-triphos-
phate (ECTP), it strongly inhibits RNA synthesis
through inhibition of RNA polymerases.2 This
mechanism is different from that of other known anti-
tumor antimetabolites, such as ara-C, 5-FU or gemci-
tabine. Although ECyd is expected to be an efficient
antitumor drug because of its remarkable antitumor
effects in experimental models, ECyd is however some-
what toxic to rapidly growing normal host cells. If
ECyd distributes selectively to tumor tissues, the toxi-
city against the normal cells would be decreased. Con-
sequently, a prodrug of ECyd, activated selectively in
tumor tissues, is a very attractive alternative.
A number of antitumor agents are widely used today in
cancer chemotherapy. However, serious problems limit-
ing their antitumor effect still exist. If an antitumor
agent could be selectively distributed to tumor tissues
via its prodrug, a masked form of the drug, toxicities
against normal tissues could then be decreased and
many of these problems would possibly be eliminated,
or at least minimized.
In recent years, we have synthesized a variety of sugar-
modified nucleoside analogues and have found 1-(3-C-
ethynyl-b-d-ribo-pentofuranosyl)cytosine (ECyd, 1, Fig.
yThis report constitutes part 220 of Nucleosides and Nucleotides: Part
219: Sukeda, M.; Ichikawa, S.; Matsuda, A.; Shuto, S. J. Org. Chem.
in press.
It is known that tumor tissues have properties different
from normal tissues. One such characteristic observed in
*Corresponding author. Tel.: +81-11-706-3228; fax: +81-11-706-
4980; e-mail: matuda@pharm.hokudai.ac.jp
0968-0896/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0968-0896(03)00104-4

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M. Nomura et al. / Bioorg. Med. Chem. 11 (2003) 2453–2461
Fig 1. ECyd and its potential prodrugs.
tumor tissues, particularly in solid tumors, is hypoxia,
which often decreases the response to chemotherapeutic
agents and radioionizing therapy. Under hypoxic con-
ditions, bio-reductive reactions would be promoted,3
therefore much effort has been devoted to developing
antitumor prodrugs which can be activated selectively in
tumor tissues with bio-reductive potency. For example,
nitrophenyl and quinone derivatives are known to be
activated under bio-reductive conditions.4,5
hydrolysis to release ECyd via the bio-reduction–
hydrolysis mechanism.
Results and Discussion
Synthesis of the benzylidene derivatives of ECyd
In addition to the target compounds 2, 3 and 6–10
described above, we also planned to synthesize the cor-
responding amino congeners 4, 5 and 11–15 (Fig. 1), the
probable reduction products of 2, 3 and 6–10, to deter-
mine if they could be readily hydrolyzed.
Recently, Threadgill and co-workers proposed a poten-
tial bio-reductively activated prodrug system using the
cyclic acetal of 5-nitrofuranylaldehyde for diol-contain-
ing drugs.6 They hypothesized that the nitro group
could be reduced to an amino group in vivo, and that
the resulting 5-aminofuranyl compound would be read-
ily hydrolyzed because of the electron-donating effect of
the amino group, which produces the deprotected drug
having a diol moiety. They confirmed that the 5-nitro-
furan-2-ylmethylidene group was hydrolyzed via reduc-
tion by a chemical reductant system such as NaBH4/Pd-C
using 1,2-dihydroxyphenylethane as a diol model; how-
ever, its bio-reduction has not been examined. Such a
cyclic acetal system may be suitable for developing
effective prodrugs of ECyd, since it has a cis-diol moiety
in the molecule. Therefore, we designed 20,30-O-(4-
nitrobenzylidene)ECyd (2) and its regio-isomer 20,30-O-
(3-nitrobenzylidene)ECyd (3) as potential prodrugs
producing ECyd in vivo selectively in tumor tissues via
the bio-reduction–hydrolysis mechanism. We also
designed the acyclic acetal derivatives 6–10, bearing an
(alkoxy)(nitrophenyl)methyl group at the 50-hydroxyl of
ECyd (Fig. 1). These acyclic acetals were also expected
to produce ECyd in vivo via the bio-reduction–hydro-
lysis mechanism. We speculated that the 4-nitrophenyl
group would be more suitable than the 3-nitrophenyl
group in the bio-reduction-hydrolysis strategy, since the
bio-reduced 4-amino moiety is able to donate electrons
by resonance thus promoting the hydrolysis as shown in
Scheme 1.
The synthesis of the 20,30-O-benzylidene derivatives of
ECyd 2–5 is shown in Scheme 2. Successive treatment of
ECyd (1) with dimethoxypropane/HClO4 in acetone and
BzCl/DMAP/Et3N in MeCN gave the fully protected
ECyd derivative 16. Removal of the isopropylidene group
of 16 with aqueous 90% TFA gave the diol 17, which was
heated with 3- or 4-nitrobenzaldehyde dimethyl acetal in
the presence of H2SO4 under reduced pressure to give the
corresponding 20,30-O-nitrobenzylidene derivatives 18 and
19, respectively. Reduction of the nitro groups of 18
and 19 was performed with Na2S2O4 in the presence of
the electron-phase transfer catalyst 227 to give the
aminobenzylidene derivatives 20 and 21, respectively.
Treatment of the benzylidene derivatives 18, 19, 20 and
21, with NaOMe/MeOH afforded the corresponding
target compounds, 2, 3, 4 and 5, respectively. The
stereochemistry of the benzylidene moiety was con-
firmed as endo by the nOe experiment of 2, as shown in
Figure 1.
The synthesis of the acyclic acetal derivatives of ECyd
6–15 is shown in Scheme 3. ECyd was treated with 3- or
4-nitrobenzaldehyde dialkyl acetal and H2SO4 in
DMSO under reduced pressure at room temperature to
50 ꢀC to afford the corresponding acyclic acetal deriva-
tives 6–10. Reduction of the nitro group was performed
using the same procedure as that for the synthesis of 4
and 5 to afford the 50-O-(alkoxy)(aminophenyl)methyl
derivatives 11–15.
In this report, we describe the synthesis of these cyclic
and acyclic acetal derivatives of ECyd and also their

M. Nomura et al. / Bioorg. Med. Chem. 11 (2003) 2453–2461
2455
Stability of the acetal derivatives of ECyd in aqueous
solution
It is recognized that tumor tissues are often more acidic
than normal tissues. The pH values of tumor tissues are
usually between 6.5 and 7.0,8 and sometimes lower
(about 6.0).8a,8c Since acetals are unstable to hydrolysis
under acidic conditions, the designed prodrugs may be
hydrolyzed selectively in acidic tumor tissues to release
ECyd. Therefore, it would be important to estimate the
effect of the pH medium on the cleavage process of the
potential prodrugs of ECyd. Two phosphate buffers,
one at pH 6.5 as a model for acidic tumor tissues and
one at the physiological pH 7.4, were used for the eval-
uations. The substrates were incubated at 37 ꢀC in these
buffers, and the time course of ECyd production was
investigated by HPLC. The results are summarized in
Table 1.
Scheme 1. The bio-reduction-hydrolysis mechanism.
Scheme 2. Synthesis of the 20,30-O-benzylidene derivatives of ECyd. (a) (1) (CH3)2C(OCH3)2, acetone, HClO4, (2) BzCl, Et3N, DMAP, CH3CN; (b)
90% TFA–H2O, rt; (c) RCH(OCH3)2, cH2SO4, 90 ꢀC, reduced pressure; (d) MeONa, MeOH; (e) 22, Na2S2O4, K2CO3, CH2Cl2–H2O.
Scheme 3. Synthesis of the 50-acetal derivatives of ECyd. (a) 3- or 4-NO2-PhCH(OR)2, cH2SO4, rt–50 ꢀC, reduced pressure; (b) 22, Na2S2O4, K2CO3,
CH2Cl2–H2O.

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M. Nomura et al. / Bioorg. Med. Chem. 11 (2003) 2453–2461
Table 1. Stability of the acetal derivatives of ECyd in aqueous mediaa
Substrate
pH 6.5
ECyd release at 2 h (%)
pH 7.4
ECyd release at 2 h (%)
t1/2 (min)
t1/2 (min)
2
3
4
5
6
7
8
9
10
11
12
13
14
15
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
14
0
0
<1
0
0
0
0
0
0
100
100
100
3
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
74
0
0
0
0
0
0
0
0
0
72
87
100
<1
2
8.4
4.8
>1000
535
32
20
>1000
>1000
7
aThe yields were determined by HPLC analysis.
All of the nitro compounds, that is, the 20,30-O-nitro-
benzylidene derivatives 2 and 3 and 50-(alkoxy)(nitro-
phenyl)methyl derivatives 6–10, were completely
resistant to hydrolysis under both of these conditions.
The 20,30-O-(3-aminobenzylidene) derivative 5 was also
stable at both pH 6.5 and pH 7.4. The corresponding
pounds were incubated with S-9 mix in a sodium phos-
phate buffer (pH 7.4) at 37 ꢀC, and the resulting
reduction products, that is, the corresponding amino
congeners, were analyzed by HPLC.9 The results are
shown in Table 2. In the case of the acyclic acetals 6–8
having a 50-O-(alkoxy)(4-nitrophenyl)methyl group, the
corresponding reduction products were not detected,
probably because they were rapidly hydrolyzed under
the reaction conditions. However, one of the hydrolysis
products, 4-aminobenzaldehyde, derived from the
hydrolysis of the amino congeners 11–13, was detected
and its yield was measured by HPLC (Table 2).
4-aminobenzylidene derivative
4 was very slowly
hydrolyzed at pH 6.5 to form a trace of ECyd after 2 h,
although it was stable at the physiological pH 7.4. We
expected the electron-donating amino-substituent to
promote the hydrolysis when it was attached at the
resonantly effective para-position. However, the experi-
ments showed that the rate of ECyd release from the
4-aminobenzylidene derivative 4 was very low.
The nitro group of the 20,30-O-(4-nitrobenzylidene) and
-(3-nitrobenzylidene) derivatives 2 and 3 was reduced
effectively by S-9 mix to form the aminobenzylidene
congeners 4 and 5, respectively; after 3 h, compounds 4
(41%) and 5 (29%) from 2 and 3, respectively, were
detected by HPLC. The reduction product of 2 was
isolated by MCI gel column chromatography and iden-
In contrast, the acyclic acetal derivatives 11–15 were
hydrolyzed more rapidly than the cyclic benzylidene
derivatives. The 50-O-(alkoxy)(4-aminophenyl)methyl
derivatives 11–13 were hydrolyzed completely at pH 6.5
within 2 h, where the t1/2 values were 14, 8.4, and 4.8
min, respectively. Although the acyclic acetals 11–13
were also hydrolyzed at the neutral pH 7.4, the rates
were slower compared with those under the acidic con-
ditions. Thus, the acyclic acetals 11–13 seemed to be
hydrolyzed in acidic tumor tissues more effectively than
in neutral normal tissues. On the other hand, the 50-O-
(alkoxy)(3-aminophenyl)methyl derivatives 14 and 15
were not readily hydrolyzed at pH 6.5 or pH 7.4.
1
tified as 4 by comparing the H NMR and mass spectra
and retention time on HPLC with those of a chemically
synthesized authentic sample. The acyclic acetals 9 and
10 having a 50-O-(alkoxy)(3-nitrophenyl)methyl group
were reduced by S-9 mix as well, but the rates were
somewhat lower than those of the 20,30-O-nitrobenzyl-
idene derivatives; after 3 h, compounds 14 (21%) and 15
(25%) from 9 and 10, respectively, were formed. Simi-
larly, reduction of the acyclic acetals 6–8 having a 50-O-
(alkoxy)(4-nitrophenyl)methyl group to the corres-
These results suggest that the acyclic acetal derivatives
of ECyd 11–13 having a 50-O-(alkoxy)(4-aminophe-
nyl)methyl group, may release ECyd selectively in acidic
tumor tissues, if these are reductively produced from the
corresponding nitro derivatives 6–8 in vivo.
Table 2. Bio-reduction of the acetal derivatives of ECyda
Yield (%)
Substrate
Product
1 h
3 h
41
Bio-reduction of the acetal derivatives of ECyd by S-9
mix
2
3
6
7
8
9
10
4
5
19
1629
12
11
9
We next examined whether the cyclic 20,30-O-nitro-
benzylidene and the acyclic 50-O-(alkoxy)(4-nitrophe-
nyl)methyl derivatives of ECyd could be biologically
reduced using rat S-9 mix as the model bio-reductant,
which is a 9000 g supernatant of rat liver homogenate
containing P450 reductases and cofactors. The com-
p-NH2PhCHO
p-NH2PhCHO
p-NH2PhCHO
14
16
12
8
21
25
10
11
15
aThe yields were determined by HPLC analysis.

M. Nomura et al. / Bioorg. Med. Chem. 11 (2003) 2453–2461
2457
ponding amino congeners 11–13 by S-9 mix was sug-
gested, since formation of 4-aminobenzaldehyde was
observed after a 3 h reaction in 16, 12 and 8% yields,
respectively.
temperature for 5 h. After addition of MeOH (6mL),
the reaction mixture was diluted with CHCl3, washed
with H2O, dried (Na2SO4), and evaporated. The residue
was purified on a silica gel column (20–50% EtOAc in
CHCl3) and then crystallized from Et2O to give pure 16
(6.5 g, 63% as white crystals): mp 207–208 ꢀC; found: C,
65.17; H, 4.88; N, 8.00. C28H25N3O7 requires C, 65.24;
These results suggest that the cyclic and the acyclic ace-
tal derivatives are substrates of P450 and can be con-
verted into the corresponding amino congeners in vivo.
1
H, 4.89; N, 8.15%; H NMR (DMSO-d6) d 11.25 (1H,
br s, 4-NH), 8.16(1H, d, J=7.9 Hz, H-6), 7.97 (2H, d,
J=7.3 Hz, Bz), 7.86(2H, d, J=7.3 Hz, Bz), 7.45–7.65
(6H, m, Bz), 7.31 (1H, br d, J=7.9 Hz, H-5), 5.98 (1H,
d, J=2.0 Hz, H-10), 5.03 (1H, d, J=2.0 Hz, H-20), 4.57–
4.69 (3H, m, H-40, H-50), 3.99 (1H, s, 30-ethynyl), 1.48,
1.55 (each 3H, each s, 20,30-O-isopropylidene); 13C
NMR (DMSO-d6) d 167.08, 165.11, 163.32, 154.13,
144.98, 133.38, 132.90, 132.64, 129.02, 128.87, 128.60,
128.31, 115.28, 96.28, 91.30, 90.65, 84.77, 82.14, 80.69,
78.54, 63.80, 27.32, 25.93; FAB-MS m/z 516(MH +).
Conclusion
We identified the 50-O-(alkoxy)(4-nitrophenyl)methyl
derivatives 6–8 as candidate prodrugs of ECyd to be
selectively activated in tumor tissues via the bio-reduc-
tion–hydrolysis mechanism owing to such characteristic
properties as hypoxia and lower pH of these tissues. The
compounds 6–8 were reduced by rat S-9 mix to the cor-
responding amino congeners 11–13, which were hydro-
lyzed to release ECyd more effectively at pH 6.5 than at
pH 7.4. This prodrug system with an acyclic acetal of
4-nitrobenzaldehyde would be applicable to other anti-
tumor agents possessing a hydroxyl group. We are now
planning to evaluate the antitumor effect of the cyclic
and the acyclic acetal derivatives of ECyd.
4-N-Benzoyl-1-[3-C-ethynyl-5-O-benzoyl-ꢀ-D-ribo-pento-
furanosyl]cytosine (17). A solution of 6 (6.02 g, 11.7
mmol) in aqueous 90% TFA (250 mL) was stirred at
room temperature for 1 h and poured into a mixture of
CH2Cl2 (1 L) and H2O (1.2 L). The organic layer sepa-
rated was washed with saturated aqueous NaHCO3,
dried (Na2SO4), and evaporated. The residue was pur-
ified on a silica gel column (10% MeOH in CHCl3) and
then crystallized from CHCl3–Et2O to give pure 17 (4.72
g, 85% as white crystals): mp 208–209 ꢀC; found: C,
Experimental
.
General methods
62.01; H, 4.26; N, 8.59. C25H21N3O7 1/3H2O requires C,
62.37; H, 4.54; N, 8.73%; 1H NMR (DMSO-d6) d 11.27
(1H, br s, 4-NH), 8.24 (1H, d, J=7.9 Hz, H-6), 7.47–
8.00 (10H, m, Bz), 7.31 (1H, br d, J=7.9 Hz, H-5), 6.25
(1H, s, 30-OH), 6.17 (1H, d, J=5.9 Hz, 20-OH), 5.85
Melting points were measured on a Yanagimoto MP-3
micromelting point apparatus and are not corrected.
1
The H- and 13C NMR spectra were recorded on a Jeol
0
0
AL-400 (400 MHz) or Jeol JNM-EX 270 (270 MHz)
spectrometer with tetramethylsilane (0.00 ppm) as an
internal standard. Chemical shifts were reported in parts
per million (d), and signals were expressed as a s (sing-
let), d (doublet), t (triplet), q (quartet), m (multiplet), or
br (broad). Coupling constants (J) were reported in Hz.
All exchangeable protons were detected by dis-
appearance on the addition of D2O. Fast atom bom-
bardment mass spectrometry (FAB-MS) was done on a
Jeol JMS-HX110 instrument at an ionizing voltage of
70 eV. TLC was done on Merck Silica gel 60 F254 pre-
coated plates. The silica gel used for column chromato-
graphy was Merck Silica gel 60 (70–230 mesh). The NH-
silica gel used for column chromatography was Fuji
Silysia DM1020 (100–200 mesh).
(1H, d, J=4.6Hz, H-1 ), 4.65 (2H, d, J=4.6Hz, H-5 ),
0
4.34 (1H, t, J=4.6Hz, H-4 ), 4.24 (1H, dd, J=4.6Hz,
5.9 Hz, H-20), 3.68 (1H, s, 30-ethynyl); 13C NMR
(DMSO-d6) d: 167.19, 165.45, 163.15, 154.52, 145.57,
133.43, 132.94, 132.65, 129.29, 129.11, 128.73, 128.33,
95.98, 89.95, 82.86, 81.94, 78.79, 72.06, 64.66; FAB-MS
m/z 476(MH +).
4-N-Benzoyl-1-[3-C-ethynyl-2,3-O-(4-nitrobenzylidene)-
A
5-O-benzoyl-ꢀ-D-ribo-pentofuranosyl]cytosine (18).
mixture of 17 (147 mg, 0.30 mmol), 4-nitrobenzaldehyde
dimethyl acetal (1.91 g, 9.7 mmol) and cH2SO4 (16 mL)
in toluene (1 mL) was stirred at 90 ꢀC under reduced
pressure for 3.5 h. After addition of Et3N (92 mL), the
resulting mixture was partitioned between CHCl3 and
H2O, and the organic layer was dried (Na2SO4) and
evaporated. The residue was purified on a silica gel col-
umn (20–66% EtOAc in CHCl3) and then crystallized
from CHCl3–Et2O to give 18 (164 mg, 90% as a white
powder): mp 201–202 ꢀC; Found: C, 62.17; H, 4.10; N,
4-N-Benzoyl-1-(3-C-ethynyl-2,3-O-isopropylidene-5-O-
benzoyl-ꢀ-D-ribo-pentofuranosyl)cytosine (16). To a sus-
pension of ECyd1b (1, 5.34 g, 20.0 mmol) in a mixture of
acetone (150 mL) and 2,2-dimethoxypropane (4.9 mL)
was added 70% HClO4 (4.3 mL, 50 mmol). The mixture
was stirred at room temperature for 40 min. To the
resulting clear solution were added 28% NH4OH (6
mL) and evaporated. The residue was purified on a
silica gel column (33–50% MeOH in CHCl3) to give an
HClO4 salt of 1-(3-C-ethynyl-2,3-O-isopropylidene-b-d-
ribo-pentofuranosyl)cytosine, a mixture of which, Et3N
(13.9 mL), DMAP (244 mg, 2.0 mmol) and BzCl (5.8
mL, 50 mmol) in CH3CN (60 mL) was stirred at room
.
8.82. C32H24N4O9 1/2H2O requires C, 62.24; H, 4.08; N,
1
9.07%; H NMR (DMSO-d6) d 11.24 (1H, s, 4-NH),
8.33 (2H, d, J=7.6Hz, Ar), 8.24 (1H, d, J=7.3 Hz, H-
6), 7.93–7.95 (4H, m, Ar, Bz), 7.73 (2H, d, J=7.6Hz,
Bz), 7.42–7.64 (6H, m, Bz), 7.25 (1H, d, J=7.3 Hz, H-
5), 6.38 (1H, s, Ar-CH), 5.99 (1H, s, H-10), 5.21 (1H, s,
H-20), 5.04 (1H, dd, J=2.9, 4.1 Hz, H-40), 4.75 (1H, dd,
J=2.9, 12.2 Hz, H-50a), 4.67 (1H, dd, J=4.1, 12.2 Hz,
H-50b), 4.15 (1H, s, 30-ethynyl); 13C NMR (DMSO-d6) d

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M. Nomura et al. / Bioorg. Med. Chem. 11 (2003) 2453–2461
167.01, 165.00, 163.37, 154.28, 148.51, 145.41, 141.56,
133.37, 132.90, 132.63, 128.93, 128.69, 128.54, 128.32,
123.67, 104.52, 96.10, 93.29, 91.29, 84.48, 84.41, 81.76,
76.71, 64.19; FAB-MS m/z 609 (MH+).
for preparing 20, after purification on a silica gel col-
umn (5% MeOH in CHCl3): Found: C, 62.77; H, 4.31;
N, 9.01. C32H26N4O7 1/3CHCl3 requires C, 62.80; H,
.
4.29; N, 9.06%; 1H NMR (DMSO-d6) d 11.22 (1H, br s,
4-NH), 8.24 (1H, d, J=7.6Hz, H-6), 7.96(2H, d, J=7.0
Hz, Bz), 7.76(2H, d, J=7.3 Hz, Bz), 7.42–7.66 (6H, m,
Bz), 7.26(1H, d, J=7.6Hz, H-5), 7.09 (1H, dd, J=7.6,
7.9 Hz, Ar), 6.81 (1H, t, J=1.8 Hz, Ar), 6.73 (1H, d,
J=7.6, 1.8 Hz, Ar), 6.65 (1H, dd, J=7.9, 1.8 Hz, Ar),
6.03 (1H, s, Ar-CH), 5.97 (1H, d, J=2.1 Hz, H-10), 5.23
(2H, s, Ar-NH2), 5.11 (1H, d, J=2.1 Hz, H-20), 4.96
(1H, dd, J=3.6, 5.1 Hz, H-40), 4.74 (1H, dd, J=3.6,
12.2 Hz, H-50a), 4.67 (1H, dd, J=5.1, 12.2 Hz, H-50b),
4.07 (1H, s, 30-ethynyl); 13C NMR (DMSO-d6) d 167.18,
165.18, 163.48, 148.81, 145.39, 145.29, 135.43, 133.45,
133.08, 132.70, 129.07, 128.87, 128.81, 128.66, 128.40,
115.48, 114.46, 112.15, 106.68, 96.17, 93.26, 90.99,
84.63, 83.69, 81.31, 77.16, 64.21; FAB-MS m/z 579
(MH+).
4-N-Benzoyl-1-[3-C-ethynyl-2,3-O-(3-nitrobenzylidene)-5-
O-benzoyl-ꢀ-D-ribo-pentofuranosyl]cytosine (19). Com-
pound 19 (165 mg, 90% as white crystals) was prepared
from 17 (147 mg, 0.30 mmol) as described for preparing
18 using 3-nitrobenzaldehyde dimethyl acetal instead of
4-nitrobenzaldehyde dimethyl acetal, after purification
on a silica gel column (20–66% EtOAc in CHCl3) and
subsequent crystallization from MeOH: mp 111–
114 ꢀC; found: C, 62.86; H, 4.09; N, 9.13. C32H24N4O9:
1
C, 63.16; H, 3.98; N, 9.21%; H NMR (DMSO-d6) d
11.23 (1H, s, 4-NH), 8.45 (1H, d, J=1.0 Hz, Ar), 8.35
(1H, dd, J=1.0, 8.3 Hz, Ar), 8.24 (1H, d, J=7.8 Hz,
H-6), 8.12 (1H, d, J=7.8 Hz, Ar), 7.94 (2H, d, J=8.1
Hz, Bz), 7.42–7.82 (9H, m, Bz, Ar), 7.25 (1H, d, J=7.8
Hz, H-5), 6.40 (1H, s, Ar-CH), 6.00 (1H, d, J=1.7 Hz,
H-10), 5.21 (1H, d,0 J=1.7 Hz, H-20), 5.05 (1H, dd,
J=3.2, 4.6Hz, H-4 ), 4.75 (1H, dd, J=3.2, 12.0 Hz,
H-50a), 4.66 (1H, dd, J=4.6, 12.0 Hz, H-50b), 4.14
(1H, s, 30-ethynyl); 13C NMR (DMSO-d6) d 167.00,
165.00, 163.37, 154.30, 147.75, 145.44, 136.95, 133.73,
133.36, 132.92, 132.63, 130.34, 128.94, 128.68, 128.56,
128.32, 125.00, 121.85, 104.46, 96.07, 93.39, 91.29,
84.50, 84.40, 81.71, 76.77, 64.19; FAB-MS m/z 609
(MH+).
Removal of the benzoyl group (general procedure). A
mixture of a substrate (18–21) (225 mmol) and NaOMe
(1.25 M in MeOH, 0.36mL) in MeOH (7 mL) was stir-
red at room temperature for 1 h, and the reaction was
then quenched with AcOH (30 mL). The mixture was
evaporated, and the residue was purified as described
below.
1-[3-C-Ethynyl-2,3-O-(4-nitrobenzylidene)-ꢀ-D-ribo-pen-
tofuranosyl]cytosine (2). After purification of the resi-
due from the reaction of 18 on a silica gel column
developed with 10–20% MeOH in CHCl3, 2 (85 mg,
94% as a colorless solid) was obtained: mp 139–142 ꢀC;
4-N-Benzoyl-1-[3-C-ethynyl-2,3-O-(4-aminobenzylidene)-
5-O-benzoyl-ꢀ-D-ribo-pentofuranosyl]cytosine (20).
A
solution of K2CO3 (319 mg, 2.31 mmol) and Na2S2O4
(366 mg, 2.1 mmol) in H2O (2.5 mL) was added to a
mixture of 18 (305 mg, 0.50 mmol) and di(C8H7)-violo-
gen 227 (5 mg, 9.2 mmol) in a mixture of CH2Cl2 (5 mL)
and H2O (1 mL) under N2 atmosphere. The resulting
mixture was stirred at 40 ꢀC for 24 h and then poured
into CHCl3 (50 mL). The organic layer separated was
washed with H2O, dried (Na2SO4), and evaporated. The
residue was purified on a silica gel column (4% MeOH
in CHCl3) to give 20 (127 mg, 44% as a colorless
amorphous): found: C, 62.68; H, 4.34; N, 8.95.
.
Found: C, 50.07; H, 3.95; N, 12.28. C18H16N4O7
1
0.35CHCl3 requires C, 49.85; H, 3.73; N, 12.67%; H
NMR (DMSO-d6) d 8.30 (2H, d, J=7.8 Hz, Ar), 7.89
(2H, d, J=7.8 Hz, Ar), 7.66 (1H, d, J=7.3 Hz, H-6),
7.34, 7.29 (each 1H, each br s, 4-NH2), 6.34 (1H, s, Ar-
CH), 6.01 (1H, d, J=3.2 Hz, H-10), 5.79 (1H, d, J=7.3
Hz, H-5), 5.13 (1H, t, J=5.0 Hz, 50-OH), 4.97 (1H, d,
0
J=3.2 Hz, H-20), 4.27 (1H, dd, J=4.2, 5.6Hz, H-4 ),
4.08 (1H, s, 30-ethynyl), 3.75 (1H, ddd, J=4.2, 5.0, 12.0
Hz, H-50a), 3.69 (1H, ddd, J=5.0, 5.6, 12.0 Hz, H-50b);
NOE, irradiates the ortho proton of the nitrobenzyli-
dene group, observe H-10 (0.8%); 13C NMR (DMSO-d6)
d 165.59, 154.65, 148.42, 141.78, 141.19, 128.43, 123.64,
105.24, 94.63, 90.55, 89.25, 85.60, 83.67, 81.44, 77.59,
60.95; FAB-MS m/z 401 (MH+).
.
C32H26N4O7 1/3CHCl3 requires C, 62.80; H, 4.29; N,
1
9.06%; H NMR (DMSO-d6) d 11.22 (1H, br s, 4-NH),
8.22 (1H, d, J=7.3 Hz, H-6), 7.95 (2H, d, J=7.6Hz,
Bz), 7.76(2H, d, J=7.6 Hz, Bz), 7.41–7.65 (6H, m, Bz),
7.26(1H, d, J=7.3 Hz, H-5), 7.24 (2H, d, J=8.6Hz,
Ar), 6.58 (2H, d, J=8.6Hz, Ar), 6.01 (1H, s, Ar-C H),
5.99 (1H, d, J=2.3 Hz, H-10), 5.37 (2H, s, Ar-NH2),
5.04 (1H, d, J=2.3 Hz, H-20), 4.92 (1H, dd, J=3.6, 5.3
Hz, H-40), 4.72 (1H, dd, J=3.6, 12.2 Hz, H-50a), 4.64
(1H, dd, J=5.3, 12.2 Hz, H-50b), 4.03 (1H, s, 30-eth-
ynyl); 13C NMR (DMSO-d6) ꢀ 166.95, 164.97, 163.21,
154.13, 150.35, 145.29, 133.26, 132.88, 132.53, 128.95,
128.69, 128.49, 128.28, 128.23, 120.97, 113.00, 107.22,
96.11, 93.00, 90.52, 84.37, 83.20, 81.02, 77.42, 64.14;
FAB-MS m/z 579 (MH+).
1-[3-C-Ethynyl-2,3-O-(3-nitrobenzylidene)-ꢀ-D-ribo-pen-
tofuranosyl]cytosine (3). After purification of the resi-
due from the reaction of 19 on a silica gel column
developed with 10–20% MeOH in CHCl3, 3 (64 mg,
94% as a colorless solid) was obtained: mp 115–120 ꢀC;
.
found: C, 48.67; H, 3.71; N, 11.92. C18H16N4O7 1/2H2O
requires C, 48.30; H, 3.62; N, 12.18%; 1H NMR
(DMSO-d6) d 8.32–8.41 (2H, m, Ar), 8.08 (1H, d, J=7.3
Hz, Ar), 7.78 (1H, t, J=7.9 Hz, Ar), 7.66 (1H, d, J=7.6
Hz, H-6), 7.31, 7.26 (each 1H, each br s, 4-NH2), 6.36
(1H, s, Ar-CH), 6.01 (1H, d, J=3.3 Hz, H-10), 5.78 (1H,
4-N-Benzoyl-1-[3-C-ethynyl-2,3-O-(3-aminobenzylidene)-
5-O-benzoyl-ꢀ-D-ribo-pentofuranosyl]cytosine (21). Com-
pound 21 (178 mg, 62% as a colorless amorphous solid)
was prepared from 19 (305 mg, 0.50 mmol) as described
0
d, J=7.6Hz, H-5), 5.10 (1H, br s, 5 -OH), 4.96(1H, d,
J=3.3 Hz, H-20), 4.28 (1H, dd, J=4.0, 6.6 Hz, H-40),
0
4.06(1H, s, 3 -ethynyl), 3.76(1H, dd, J=4.0, 11.9 Hz,

M. Nomura et al. / Bioorg. Med. Chem. 11 (2003) 2453–2461
2459
H-50a), 3.69 (1H, dd, J=6.6, 11.9 Hz, H-50b); 13C NMR
(DMSO-d6) d 165.60, 154.66, 147.72, 141.21, 137.11,
133.61, 130.32, 124.90, 121.76, 105.22, 94.60, 90.61,
89.35, 85.68, 83.61, 81.40, 77.65, 60.95; FAB-MS m/z
401 (MH+).
C, 51.75; H, 4.52; N, 12.65. C19H21N4O8 1/2H2O
requires C, 51.70; H, 4.80; N, 12.69%; 1H NMR
(DMSO-d6) d 8.23–8.28 (2H, m, Ar), 7.66–7.71 (2.6H,
m, Ar, H-6), 7.55 (0.4H, J=7.3 Hz, H-6), 7.18 (2H, br s,
4-NH2), 5.95 (1H, br s, 30-OH), 5.81–5.88 (2H, m, H-10,
20-OH), 5.68–5.70 (1.6H, m, H-5, PhCH), 5.55 (0.4H, d,
J=7.3 Hz, H-5), 4.00–4.10 (2H, m, H-20, H-40), 3.68–
3.86(2H, m, H-5 0), 3.58 (0.4H, s, 30-ethynyl), 3.57 (0.6H,
s, 30-ethynyl), 3.32 (3H, s, CH3O); FAB-MS m/z 433
(MH+).
.
1-[3-C-Ethynyl-2,3-O-(4-aminobenzylidene)-ꢀ-D-ribo-pen-
tofuranosyl]cytosine (4). The residue from the reaction
of 20 was partitioned between H2O and CHCl3, and the
aqueous layer was evaporated and purified on an ODS
column (0–15% MeCN in H2O) to give 4 (68 mg, 81%
as a colorless solid): Found: C, 55.95; H, 4.93; N, 14.44.
1-[3-C-Ethynyl-5-O-[1-ethoxy-1-(4-nitrophenyl)methyl]-
ꢀ-D-ribo-pentofuranosyl]cytosine (7). Compound 7 (755
mg, 23%) was obtained as a colorless foam: Found: C,
.
C18H18N4O5 H2O requires C, 55.67; H, 5.19; N,
14.43%; 1H NMR (DMSO-d6) d 7.63 (1H, d, J=7.4 Hz,
H-6), 7.32, 7.26 (each 1H, each br s, 4-NH2), 7.20 (2H,
d, J=7.8 Hz, Ar), 6.57 (2H, d, J=7.8 Hz, Ar), 6.02 (1H,
d, J=3.1 Hz, H-10), 5.98 (1H, s, Ar-CH), 5.79 (1H, d,
J=7.4 Hz, H-5), 5.35 (2H, br s, Ar-NH2), 5.07 (1H, br,
50-OH), 4.77 (1H, d, J=3.1 Hz, H-20), 4.18 (1H, dd,
.
51.38; H, 5.12; N, 11.89. C20H22N4O8 H2O requires C,
51.72; H, 5.21; N, 12.06%; 1H NMR (DMSO-d6) d
8.23–8.27 (2H, m, Ar), 7.66–7.71 (2.7H, m, Ar, H-6),
7.58 (0.3H, J=7.3 Hz, H-6), 7.19 (2H, br s, 4-NH2),
5.95 (1H, br s, 30-OH), 5.80–5.88 (2H, m, H-10, 20-OH),
5.76(0.3H, s, PhC H), 5.73 (0.7H, s, PhCH), 5.68 (0.3H,
d, J=7.3 Hz, H-5), 5.66 (0.7H, d, J=7.6Hz, H-5),
4.01–4.10 (2H, m, H-20, H-40), 3.78–3.82 (1.4H, m, H-
50), 3.59–3.72 (2.6H, m, H-50, CH3CH2O), 3.57 (0.3H, s,
0
J=4.0, 6.8 Hz, H-40), 3.96(1H, s, 3 -ethynyl), 3.74 (1H,
dd, J=4.0, 12.0 Hz, H-50a), 3.69 (1H, dd, J=6.8, 12.0
Hz, H-50b); 13C NMR (DMSO-d6) d 165.67, 154.75,
150.46, 141.22, 128.35, 121.32, 113.12, 108.11, 94.71,
90.02, 88.99, 85.77, 82.41, 80.73, 78.38, 61.05; FAB-MS
m/z 371 (MH+).
0
30-ethynyl), 3.56(0.7H, s, 3 -ethynyl), 1.13-1.20 (3H, m,
CH3CH2O); FAB-MS m/z 447 (MH+).
1-[3-C-Ethynyl-2,3-O-(3-aminobenzylidene)-ꢀ-D-ribo-pen-
tofuranosyl]cytosine (5). The residue from the reaction
of 21 was partitioned between H2O and CHCl3, and the
aqueous layer was evaporated and purified on an ODS
column (0–15% MeCN in H2O) to give 5 (74 mg, 89%
as a colorless solid): found: C, 55.86; H, 4.93; N, 14.36.
1-[3-C-Ethynyl-5-O-[1-isopropoxy-1-(4-nitrophenyl)me-
thyl]-ꢀ-D-ribo-pentofuranosyl]cytosine (8). Compound 8
(755 mg, 22%) was obtained as a colorless foam:
.
Found: C, 52.52; H, 5.22; N, 11.51. C21H24N4O8 H2O
requires C, 52.72; H, 5.48; N, 11.71%; 1H NMR
(DMSO-d6) d 8.23–8.27 (2H, m, Ar), 7.61–7.71 (2.7H,
m, Ar, H-6), 7.58 (0.3H, J=7.6Hz, H-6), 7.19 (2H, br s,
4-NH2), 5.92 (1H, br s, 30-OH), 5.78–5.89 (3H, m, H-10,
20-OH, PhCH), 5.67 (0.5H, d, J=7.6Hz, H-5), 5.06
(0.5H, d, J=7.6Hz, H-5), 3.94–4.10 (3H, m, H-2 0, H-40,
(CH3)2CHO), 3.73–3.75 (1H, m, H-50), 3.60–3.66 (1H,
.
C18H18N4O5 H2O requires C, 55.67; H, 5.19; N,
14.43%; 1H NMR (DMSO-d6) d 7.56(1H, d, J=7.6Hz,
H-6), 7.26, 7.22 (each 1H, each br s, 4-NH2), 6.99 (1H,
dd, J=7.6, 7.9 Hz, Ar), 6.69 (1H, br, Ar), 6.60 (1H, d,
J=7.6Hz, Ar), 6.56(1H, d, J=7.9 Hz, Ar), 5.95 (1H, d,
J=3.0 Hz, H-10), 5.93 (1H, s, Ar-CH), 5.73 (1H, d,
0
m, H-50), 3.57 (0.5H, s, 30-ethynyl), 3.56(0.5H, s, 3 -
J=7.6Hz, H-5), 5.14 (2H, s, Ar-NH ), 5.04 (1H, br s,
ethynyl), 1.12–1.22 (6H, m, (CH3)2CHO); FAB-MS m/z
461 (MH+).
2
50-OH), 4.78 (1H, d, J=3.0 Hz, H-20), 4.12 (1H, dd,
J=4.0, 6.6 Hz, H-40), 3.94 (1H, s, 30-ethynyl), 3.68 (1H,
dd, J=4.0, 11.9 Hz, H-50a), 3.61 (1H, dd, J=6.6, 11.9
Hz, H-50b); 13C NMR (DMSO-d6) d 165.47, 154.52,
148.57, 140.98, 135.39, 128.66, 115.23, 114.23, 111.91,
107.42, 94.60, 90.37, 88.98, 85.85, 82.60, 80.90, 78.03,
60.95; FAB-MS m/z 371 (MH+).
1-[3-C-Ethynyl-5-O-[1-methoxy-1-(3-nitrophenyl)me-
thyl]-ꢀ-D-ribo-pentofuranosyl]cytosine (9). Compound 9
(980 mg, 30%) was obtained as a colorless foam:
.
Found: C, 50.04; H, 4.24; N, 12.05. C19H21N4O8 1/
4CHCl3 requires C, 50.02; H, 4.42; N, 12.12%; 1H
NMR (DMSO-d6) d 8.21–8.25 (2H, m, Ar), 7.85–7.89
(1H, m, Ar), 7.67–7.74 (1.7H, m, Ar, H-6), 7.58 (0.3H,
J=7.6Hz, H-6), 7.21, 7.17 (each 1H, br s, 4-NH 2), 5.95
(1H, s, 30-OH), 5.83–5.95 (2H, m, H-10, 20-OH), 5.78
(0.3H, s, PhCH), 5.76(0.7H, s, PhC H), 5.69 (0.7H, d,
J=7.6Hz, H-5), 5.57 (0.3H, d, J=7.6Hz, H-5), 4.00–
4.10 (2H, m, H-20, H-40), 3.57–3.80 (4H, m, H-50,
CH3CH2O), 3.54 (0.7H, s, 30-ethynyl), 3.53 (0.3H, s, 30-
ethynyl), 1.14–1.21 (3H, m, CH3CH2O); FAB-MS m/z
433 (MH+).
Synthesis of the acyclic acetal derivatives 6–10 (general
procedure). A mixture of 1 (2.0 g, 7.5 mmol), the
corresponding nitrobenzaldehyde dialkyl acetal (10
mmol), and cH2SO4 (600 mL, 11.3 mmol) in DMSO (9
mL) was stirred at ambient temperature under reduced
pressure for 6h, and then Et N (4.74 mL) was added.
3
The mixture was poured into H2O (20 mL), and the
resulting mixture was extracted with EtOAc. The
organic layer was washed with H2O, dried (Na2SO4),
and evaporated. The residue was purified on a silica gel
column (10–20% MeOH in CHCl3) to give the corres-
ponding acyclic acetal.
1-[3-C-Ethynyl-5-O-[1-ethoxy-1-(3-nitrophenyl)methyl]-
ꢀ-D-ribo-pentofuranosyl]cytosine (10). Compound 10
(1.02 g, 31%) was obtained as a colorless foam: found:
.
1-[3-C-Ethynyl-5-O-[1-methoxy-1-(4-nitrophenyl)me-
thyl]-ꢀ-D-ribo-pentofuranosyl]cytosine (6). Compound 6
(996mg, 31%) was obtained as a colorless foam: found:
C, 52.76; H, 4.96; N, 12.26. C20H22N4O8 1/2H2O
requires C, 52.75; H, 5.09; N, 12.30%; 1H NMR
(DMSO-d6) d 8.21–8.25 (2H, m, Ar), 7.84–7.89 (1H, m,

2460
M. Nomura et al. / Bioorg. Med. Chem. 11 (2003) 2453–2461
Ar), 7.66–7.74 (1.5H, m, Ar, H-6), 7.56 (0.5H, J=7.3
Hz, H-6), 7.19, 7.16 (each 1H, br s, 4-NH2), 5.95 (1H, s,
30-OH), 5.83–5.95 (2H, m, H-10, 20-OH), 5.68–5.71
(1.5H, m, PhCH, H-5), 5.56(0.5H, d, J=7.3 Hz, H-5),
4.01–4.10 (2H, m, H-20, H-40), 3.67–3.86 (2H, m, H-50),
3.55 (0.5H, s, 30-ethynyl), 3.54 (0.5H, s, 30-ethynyl), 3.33
(3H, m, CH3O); FAB-MS m/z 447 (MH+).
(1H,d, J=8.3 Hz, Ph), 6.52 (1H, d, J=8.3 Hz, Ph), 6.51
(1H, d, J=8.3 Hz, Ph), 5.89 (0.5H, d, J=6.6 Hz, H-10),
5.87 (0.5H, d, J=6.6 Hz, H-10), 5.79 (2H, br, 20-OH, 30-
OH), 5.66 (0.5H, d, J=7.3 Hz, H-5), 5.51 (0.5H, d, J=7.3
Hz, H-5), 5.43 (0.5H, m, PhCH), 5.37 (0.5H, m, PhCH),
5.09 (2H, s, PhNH2), 3.56–4.08 (5H, m, H-20, H-40, H-50,
(CH3)2CHO), 3.54 (0.5H, s, 30-ethynyl), 3.53 (0.5H, s, 30-
ethynyl),1.05–1.22 (6H, m, (CH3)2CHO); FAB-MS
(negative) m/z 429 [(MꢂH)ꢂ]; FAB-HRMS (negative)
429.1794 [(MꢂH)ꢂ, C21H25N4O6 requires 429.1774].
Reduction of the acyclic acetals 6–10 (general proce-
dure). A solution of K2CO3 (120 mg, 0.87 mmol) and
Na2S2O4 (140 mg, 0.81 mmol) in H2O (1 mL) was added
to a mixture of a substrate (6–10, 0.1 mmol)) and
di(C8H7)-viologen 227 (2 mg, 3.8 mmol) in a mixture of
CH2Cl2 (2 mL) and H2O (0.1 mL) under N2 atmos-
phere. The resulting mixture was stirred at room tem-
perature for 2 h and then partitioned between CH2Cl2
and H2O. After washing with CH2Cl2 (3ꢁ), the aqueous
layer was saturated with NaCl and then extracted with
THF (4ꢁ). The combined THF layer was dried
(Na2SO4) and evaporated. The residue was purified on a
NH-silica gel column (15% MeOH in CHCl3) to give
the corresponding reduction product.
1-[3-C-Ethynyl-5-O-[1-methoxy-1-(3-aminophenyl)me-
thyl]-ꢀ-D-ribo-pentofuranosyl]cytosine (14). Compound
14 (2.9 mg, 7.2%) was obtained as a colorless amor-
1
phous: H NMR (DMSO-d6) d 7.72 (0.5H, d, J=7.6
Hz, H-6), 7.59 (0.5H, J=7.6Hz, H-6), 7.18 (2H, br s, 4-
NH2), 6.97–7.04 (1H, m, Ph), 6.61 (1H, s, Ph), 6.50–6.54
(2H, d, Ph), 5.93 (1H, s, 30-OH), 5.89 (0.5H, d, J=6.3
Hz, H-10), 5.87 (0.5H, d, J=6.3 Hz, H-10), 5.81 (1H, br,
20-OH), 5.68 (0.5H, d, J=7.6Hz, H-5), 5.51 (0.5H, d,
J=7.6Hz, H-5), 5.35 (0.5H, m, PhC H), 5.31 (0.5H, m,
PhCH), 5.07 (2H, s, PhNH2), 3.99–4.09 (2H, m, H-20,
H-40), 3.56–3.80 (2H, m, H-50), 3.55 (1H, s, 30-ethynyl),
3.22 (1.5H, s, CH3O). 3.21 (1.5H, s, CH3O); FAB-MS
(negative) m/z 401 [(MꢂH)ꢂ]; FAB-HRMS (negative)
401.1495 [(MꢂH)ꢂ, C19H21N4O6 requires 401.1461].
1-[3-C-Ethynyl-5-O-[1-methoxy-1-(4-aminophenyl)me-
thyl]-ꢀ-D-ribo-pentofuranosyl]cytosine (11). Compound
11 (3 mg, 7.6%) was obtained as a colorless amorphous:
1H NMR (DMSO-d6) d 7.74 (0.6H, d, J=7.3 Hz, H-6),
7.60 (0.4H, J=7.3 Hz, H-6), 7.19 (2H, br s, 4-NH2),
7.04 (0.8H,d, J=8.3 Hz, Ph), 7.03 (1.2H, d, J=8.3 Hz,
Ph), 6.53 (0.8H, d, J=8.3 Hz, Ph), 6.52 (1.2H, d, J=8.3
Hz, Ph), 5.90 (0.4H, d, J=6.3 Hz, H-10), 5.88 (0.6H, d,
J=6.3 Hz, H-10), 5.80 (2H, br s, 20-OH, 30-OH), 5.67
(0.6H, d, J=7.3 Hz, H-5), 5.54 (0.4H, d, J=7.3 Hz,
H-5), 5.38 (0.6H, m, PhCH), 5.34 (0.4H, m, PhCH),
5.12 (2H, s, PhNH2), 3.99–4.09 (2H, m, H-20, H-40),
3.57–3.74 (2H, m, H-50), 3.54 (1H, s, 30-ethynyl), 3.45–
3.52 (2H, m, CH3CH2O), 1.11 (3H, q, J=6.6 Hz,
CH3CH2O); FAB-MS (negative) m/z 401 [(MꢂH)ꢂ];
FAB-HRMS (negative) 401.1481 [(MꢂH)ꢂ, C19H21N4O6
requires m/z 401.1461].
1-[3-C-Ethynyl-5-O-[1-ethoxy-1-(3-aminophenyl)methyl]-
ꢀ-D-ribo-pentofuranosyl]cytosine (15). Compound 15
(3.3 mg, 8.0%) was obtained as a colorless amorphous:
1H NMR (DMSO-d6) d 7.72 (0.6H, d, J=7.6Hz, H-6),
7.62 (0.4H, J=7.6Hz, H-6), 7.17 (2H, br s, 4-NH 2),
6.96–7.03 (1H,m, Ph), 6.49–6.63 (3H, d, Ph), 5.91 (1H,
s, 30-OH), 5.88 (0.4H, d, J=6.3 Hz, H-10), 5.87 (0.6H, d,
J=6.3 Hz, H-10), 5.80 (1H, br s, 20-OH), 5.67 (0.6H, d,
J=7.6Hz, H-5), 5.55 (0.4H, d, J=7.6Hz, H-5), 5.40
(0.6H, m, PhCH), 5.36(0.4H, m, PhC H), 5.06(2H, s,
PhNH2), 3.98–3.98 (2H, m, H-20, H-40), 3.57–3.74 (2H,
m, H-50), 3.56(0.6H, s, 3 0-ethynyl), 3.54 (0.4H, s, 30-
ethynyl), 3.45–3.54 (2H, m, CH3CH2O), 1.04–1.16(3H,
m, CH3CH2O); FAB-MS m/z 455 [(M+K)+]; FAB-
HRMS 455.1338 [(M+K)+, C20H24N4O6K requires
455.1333].
1-[3-C-Ethynyl-5-O-[1-ethoxy-1-(4-aminophenyl)methyl]-
ꢀ-D-ribo-pentofuranosyl]cytosine (12). Compound 12
(3.7 mg, 8.9%) was obtained as a colorless amorphous:
1H NMR (DMSO-d6) d 7.74 (0.5H, d, J=7.6Hz, H-6),
7.65 (0.5H, J=7.6Hz, H-6), 7.20, 7.16(each 1H, each
br s, 4-NH2), 7.04 (2H,d, J=8.3 Hz, Ph), 6.53 (1H, d,
J=8.3 Hz, Ph), 6.52 (1H, d, J=8.3 Hz, Ph), 5.90 (0.5H,
d, J=6.6 Hz, H-10), 5.89 (0.5H, d, J=6.6 Hz, H-10),
5.85 (2H, br s, 20-OH, 30-OH), 5.66 (0.5H, d, J=7.3 Hz,
H-5), 5.51 (0.5H, d, J=7.3 Hz, H-5), 5.33 (0.5H, m,
PhCH), 5.28 (0.5H, m, PhCH), 5.12 (2H, s, PhNH2),
3.99–4.09 (2H, m, H-20, H-40), 3.62–3.77 (2H, m, H-50),
3.54 (1H, s, 30-ethynyl), 3.19 (3H, s, CH3O); FAB-MS
(negative) m/z 415 [(MꢂH)ꢂ]; FAB-HRMS (negative)
415.1623 [(MꢂH)ꢂ, C20H23N4O6 requires 415.1618].
Stability of the acetal derivatives of ECyd in aqueous so-
lution. An aqueous cytidine solution (0.50 mM, 50 mL)
was added to a potassium phosphate buffer (200 mM,
pH 6.0 or 7.4, 900 mL) as an internal standard and the
solution was preincubated at 37 ꢀC. To the solution was
added a solution of a substrate (2.0 mm in DMSO, 50
mL), and the resulting mixture was kept at 37 ꢀC and
analyzed by HPLC. HPLC conditions for 2–5: column,
Inertsil ODS-2 5 mm 4.6ꢁ150 mm; eluent A, 2%
CH3CN in potassium phosphate buffer (50 mM, pH
7.0); eluent B, 30% CH3CN in potassium phosphate
buffer (50 mM, pH 7.0); gradient, 0–8 min, A 100%, 15–
35 min, B 100%; flow rate, 1.0 mL/min; detection, UV
(280 nm). Internal standard (cytidine) was eluted at 2.8
min, ECyd at 4.5 min, 2 at 26.0 min, 3 at 25.5 min, 4 at
21.2 min, and 5 at 21.7 min. HPLC conditions for 9–15:
column, Inertsil ODS-2 5 mm 4.6ꢁ150 mm; eluent A,
2% CH3CN in potassium phosphate buffer (50 mM, pH
7.0); eluent B, 40% CH3CN in potassium phosphate
1-[3-C-Ethynyl-5-O-[1-isopropoxy-1-(4-aminophenyl)me-
thyl]-ꢀ-D-ribo-pentofuranosyl]cytosine (13). Compound
13 (1.7 mg, 4.0%) was obtained as a colorless amor-
1
phous: H NMR (DMSO-d6) d 7.74 (0.5H, d, J=7.3
Hz, H-6), 7.72 (0.5H, J=7.3 Hz, H-6), 7.19, 7.15 (each
1H, each br s, 4-NH2), 7.05 (1H,d, J=8.3 Hz, Ph), 7.04

M. Nomura et al. / Bioorg. Med. Chem. 11 (2003) 2453–2461
2461
buffer (50 mM, pH 7.0); gradient, 0–8 min, A 100%, 15–
35 min, B 100%; flow rate, 1.0 mL/min; detection, UV
(280 nm). Internal standard (cytidine) was eluted at 2.8
min, ECyd at 4.5 min, 6 at 20.0 min, 7 at 21.3 min, 8 at
22.8 and 22.9 min, 9 at 19.9 min, 10 at 21.1 min, 11 at 17.7
min, 12 at 18.6min, 13 at 19.4 and 19.5 min, 14 at
18.0 min, and 15 at 18.9 min.
Chem. 1996, 39, 5005. (c) Hattori, H.; Nozawa, E.; Iino, T.;
Yoshimura, Y.; Shuto, S.; Shimamoto, Y.; Nomura, M.;
Fukushima, M.; Tanaka, M.; Sasaki, T.; Matsuda, A. J. Med.
Chem. 1998, 41, 2892.
2. (a) Tabata, S.; Tanaka, M.; Matsuda, A.; Fukushima, M.;
Sasaki, T. Oncology Rep. 1996, 3, 1029. (b) Tabata, S.; Tanaka,
M.; Endo, Y.; Obata, T.; Matsuda, A.; Sasaki, T. Cancer Lett.
1997, 116, 225. (c) Takatori, S.; Tsutsumi, S.; Hidaka, M.;
Kanda, H.; Matsuda, A.; Fukushima, M.; Wataya, Y.
Nucleosides Nucleotides 1998, 17, 1309. (d) Matsuda, A.;
Fukushima, M.; Wataya, Y.; Sasaki, T. Nucleosides Nucleo-
tides 1999, 18, 811. (e) Takatori, S.; Kanda, H.; Takenaka, K.;
Wataya, Y.; Matsuda, A.; Fukushima, M.; Shimamoto, Y.;
Tanaka, M.; Sasaki, T. Cancer Chemother. Pharmacol. 1999, 44,
97. (f) Shimamoto, Y.; Fujioka, A.; Kazuno, H.; Murakami,
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ima, M. Jpn. J. Cancer Res. 2001, 92, 343. (g) Shimamoto, Y.;
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93, 445. (h) Shimamoto, Y.; Koizumi, K.; Okabe, H.; Kazuno,
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4. (a) Jenkins, T. C.; Nalor, M. A.; O’Neill, P.; Threadgill,
M. D.; Colls, S.; Stratford, I. J.; Adams, G. E.; Fielden, E. M.;
Suto, M. J.; Steir, M. J. J. Med. Chem. 1990, 33, 2603. (b)
Mulcahy, R. T.; Gipp, J. J.; Schmidt, J. P.; Joswig, C.; Borch,
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Reduction of the acetal derivatives of ECyd by S-9 mix.
A mixture of a cofactor solution (0.9 mL) described
below and an aqueous substrate solution (2.0 mM, 0.1
mL) was preincubated at 37 ꢀC for 5 min. To the solu-
tion was added rat liver S-9 (WAKO, phenobarbital and
5,6-benzoflavone induced, 0.1 mL), and the mixture was
kept at 37ꢀC. The reaction was quenched by an addition
of CH3CN (3 mL) at 0.5, 1 and 3 h. The resulting mixture
was centrifuged (6400 rpm, 6 min), and the supernatant
(1.2 mL) was dried under N2 flow at 50 ꢀC. To the residue
was added sodium phosphate buffer (200 mM, pH, 6.0,
0.3 mL), and the solution was filtrated through Millipore
Ultrafree C3LCC by centrifuging. The filtrate was ana-
lyzed by HPLC under the conditions described above.
Concentration of the cofactor solution: MgCl2, 8.9 mM;
KCl, 37 mM; G-6-P, 5.6 mM; NADPH, 4.4 mM; NADH,
4.4 mM; sodium phosphate buffer (pH 7.4), 11 mM.
Isolation of the product 4 by the S-9 mix reduction of 2.
A mixture of a cofactor solution (45 mL) described
above, an aqueous solution of 2 (2.0 mM, 5 mL, 10
mmol) and rat liver S-9 (WAKO, phenobarbital and
5,6-benzoflavone induced, 5 mL) was kept at 37 ꢀC for 5
h, and CH3CN (3 mL) was added. The supernatant was
evaporated, and the residue was diluted with H2O (5
mL) and filtrated through PTFE membrane filter. The
filtrate was purified on an MCI gel (CHP-20P) column
(1.5 cm I. D. ꢁ15 cm, 0–10% CH3CN in H2O) to give 4,
5. (a) Tomasz, M.; Lipman, R.; Chowdary, D.; Pawlak, J.;
Verdine, G. L.; Nakanishi, K. Science 1987, 235, 1204. (b)
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119, 5999. (d) Nayler, M. A.; Swann, E.; Everett, S. A.; Jaffar,
M.; Nolan, J.; Robertson, N.; Lockyer, S. D.; Patel, K. B.;
Dennis, M. F.; Stratford, M. R. L.; Wardman, P.; Adams,
G. E.; Moody, C. J.; Stratford, I. J. J. Med. Chem. 1998, 41,
2720 and references cited herein. (e) Tomasz, M.; Das, A.;
Tang, K. S.; Ford, M. G. J.; Minnock, A.; Musser, S. M.;
Waring, M. J. J. Am. Chem. Soc. 1998, 120, 11581.
1
of which H NMR and mass spectra and retention time
on HPLC were identical to those of the authentic 4.
Acknowledgements
6. (a) Mahmud, N. P.; Garrett, S. W.; Threadgill, M. D.
Anticancer Drug Des 1998, 13, 655. (b) Berry, J. M.; Watson,
C. Y.; Whish, W. J. D.; Threadgil, M. D. J. Chem. Soc., Per-
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7. Park, K. K.; Oh, C. H.; Joung, W. K. Tetrahedron Lett.
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8. (a) Vaupel, P. W.; Frinak, S.; Bicher, H. I. Cancer Res.
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This investigation was supported by a Grant-in-Aid for
Creative Scientific Research (13NP0401) from the Japan
Society for Promotion of Science. We are also grateful
to Ms. H. Matsumoto, A. Maeda, S. Oka, and N.
Hazama (Center for Instrumental Analysis, Hokkaido
University) for technical assistance with NMR, MS, and
elemental analysis.
References and Notes
9. HPLC analysis of ECyd (1), which is possible to be pro-
duced in the bio-reduction system, was unsuccessful, because
cofactors used for the bio-reduction could not be separated
from 1 on HPLC.
1. (a) Matsuda, A.; Hattori, H.; Tanaka, M.; Sasaki, T.
Bioorg. Med. Chem. Lett. 1996, 6, 1887. (b) Hattori, H.;
Tanaka, M.; Fukushima, M.; Sasaki, T.; Matsuda, A. J. Med.